This proposal requests continued support for ongoing studies of the structure, bioinorganic chemistry and allosteric regulation by zinc of selected zinc metalloregulatory transcription factors derived from mammalian and prokaryotic organisms which play roles in zinc homeostasis. Human metal-responsive element (MRE)-binding transcription factor-1 (MTF-1) possesses six Cys2-His2 zinc finger domains and activates the expression of metallothionein genes in a zinc-dependent manner. Previous studies led the PI to hypothesize that the N-terminal four fingers play a structural role in mediating high affinity binding to the MRE while the C-terminal finger domains perform a zinc sensor and/or transducer role. The following experiments on MTF-1 are proposed: 1) Define the solution structure of a C-terminal zinc finger fragment (finger domains 4-6) of MTF-1 using multi-dimensional NMR spectroscopy. These studies will be complemented by equilibrium and kinetic metal binding studies to delineate the unusual metal binding properties of these metalloregulatory domains; and 2) Test the metalloregulatory model of MTF-1 function in vivo by determining the metal-dependent transcriptional activation efficacy of broken-finger mutants of intact MTF-1 by transfection experiments in mammalian and yeast cells. Comparative studies of three evolutionarily related prokaryotic zinc metalloregulatory repressors, Synechococcus SmtB, Synechocystis ZiaR, S. aureus CzrA and a cadmium repressor, S. aureus pI258 CadC are also proposed. These studies will provide a critical test of the metalloregulatory hypothesis which holds that the biological specificity and mechanism of regulation is encoded in the bioinorganic chemistry (metal coordination chemistry, specificity, affinity and stoichiometry) and the thermodynamic linkage relationships between metal binding, DNA binding and protein self-association of these metal switch proteins. Results of the proposed studies will provide new molecular insights into the relationships between structure, coordination chemistry and regulation by metal ions in metalloregulatory transcription factors, which, over the longer term, will permit their systematic re-engineering as templates for metal-site redesign and metal sensors with novel properties, tailored to biomedical and bioremediation uses.